Feces has long been classified a potentially infectious waste product from an animal's digestive tract which is collected to test for parasites, such as pinworms and/or their eggs or to detect pathogenic bacteria and fungi in symptomatic animals and humans. Recently, however, with the rise in personalized medicine and wide-scale commercialization of pre- and pro-biotics, the diagnostic and, in particular, the prognostic value of this “waste” product has escalated. Simply a change in dietary habit has been shown to affect the microbiota or microbial community composition in feces (Walker et al, 2011; Wu et al, 2011) which, in turn, can impact health and reduce the incidence of certain diseases.
Colonization of the gastro-intestinal (GI) tract begins at birth, and the microbial community that develops over time is shaped by many influences, including the individual's genetic make-up, age, sex, nutrition, antibiotic use and other pharmaceuticals consumed, disease state, lifestyle, geographical location/environment, chemical exposure, surgical interventions and more. A diverse microbial community colonizes the intestine consisting of approximately 100 trillion bacteria which play a significant role in human health, in particular, the digestion of food, host energy metabolism, synthesis of essential vitamins, epithelium maturation, degradation of bile salts, metabolism of drugs and dietary carcinogens, as well as protecting the gut from pathogen colonization.
The ‘gut microbiome’ is the term given to describe this vast collection of symbiotic microorganisms in the human GI system and their collective interacting genomes. However, the understanding of these functional interactions between the gut microbiota and host physiology is in its infancy. The Human Microbiome Project revealed that the gut microbiome is approximately 150 times larger than the human genome, consisting of somewhere between 300 and 1000 bacterial species and more than 7000 strains. In most mammals, the gut microbiome is dominated by four bacterial phyla: Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria (Ley et al., 2007). A new area of work relates to the analysis of the interaction of the gut microbiome with gut parasites, viruses, yeasts, and numerous fungi, such as Candida, Saccharomyces, Aspergillus, and Penicillium. Some experts have suggested that the total information encoded by the human genome alone is not enough to carry out all of the body's biological functions (Lee and Mazmanian, 2010) and point to symbiosis between bacteria and humans as an explanation. With only around 10 percent of a human's cells being actually human, with microbes making up the remaining 90 percent, humans can be thought of as hosts for our microbe guests or super-organisms.
For many decades, intestinal microbes have been implicated in the initiation of colon cancer (Aries et al., 1969; Moore and Moore, 1995). More recently, Helicobacter pylori infection has been identified as a major cause of gastric (stomach) cancer, gastric lymphoma, and peptic ulcer disease (Parsonnet et al., 1991). It turns out, however, that gut microbes have more influence on how we feel and behave than we know. Due to increasing evidence that communication exists between the gut and the brain, the gut has been dubbed the ‘second brain.’ Evidence suggests that numerous diseases, such as cardiovascular disease, diabetes, stress/anxiety, autism, Crohn's disease, Irritable Bowel Disease (IBD), allergic disorders, metabolic syndrome, and neurologic inflammation may result from dysregulation of the gut microbiome. However, researchers are just beginning to decipher what is now termed the ‘microbiome-gut-brain axis’, i.e., how microorganisms colonizing the GI tract can influence biological functions beyond the gut, in particular, the molecular mechanisms or crosstalk by which the gut microbiome impacts immunological, endocrine and neurological diseases in its host (Grenham et al., 2011; Kinross et al., 2011). For instance, many microbes produce neurometabolites that are either neurotransmitters or modulators of neurotransmission, including GABA, noradrenaline, serotonin, dopamine, and acetylcholine, which act directly on nerve terminals in the gut or via enterochromaffin cells present throughout the GI tract. Carbohydrates from dietary fibre are also broken down by microbes, resulting in the production of neuroactive chemicals, such as, n-butyrate, acetate, hydrogen sulphide and propionate. In addition, microbes shed metabolites, such as proteins, carbohydrates, and other molecules, which can leave the gut and play a role in signalling disease throughout the body.
In both healthy and diseased individuals, as well as identifying the hundreds of different species making up the gut microbial community, it is critical to gain an understanding of the functionality of the consortia of bacteria as a whole. For instance, the composition of the microbiota determines competition for dietary ingredients as growth substrates, conversion of sugar into inhibitory fermentation products, production of growth substrates, release of bacteriocins (molecules toxic to other bacterial species), stimulation of the innate immune system, competition against microbes colonizing the gut wall and gut-barrier function, and more. Unfortunately, traditional microbiological culture techniques have proven largely unsuccessful in helping to determine the identity and function of members of the gut microbiome, due to significant limitations stemming from their reliance on appropriate growth nutrients and complex conditions for the entire intestinal microflora to flourish in vitro. Estimates indicate that only 20-40% (Apajalahti et al., 2003) of the total intestinal microflora can be cultivated by standard culture techniques, so the vast majority of microbial biodiversity has been missed by cultivation-based methods. This factor is further compounded by the need to ensure viability of the intestinal microflora in vitro, many of which are anaerobic (O'Sullivan, 2000).
Numerous culture media inherently select against some bacteria, in particular, ones that require extra or selective agents or bacteria in a physiological state which is not conducive to culturing directly from feces or intestinal material. Also, traditional morphological examination and biochemical tests for identifying and characterizing intestinal microflora are extremely labour-intensive, time-consuming, and lack precision, thus limiting their effectiveness for analyzing specimens from a large number of individuals and comparing the relatedness between bacterial species from different individuals. Therefore, quick methods to capture and stabilize or “snap-shot” the microbiome at the point of collection, in conjunction with culture-independent molecular tools, such as 16S ribosomal RNA gene-based approaches, TaqMan probes, digital and LATE PCR, and metagenomic sequencing, are needed to overcome these limitations and biases, so a true and detailed picture of this rich ecosystem can be revealed.
Today, approximately 1 out of every 20 hospitalized patients will contract a hospital-acquired infection (HAI). While most types of HAIs are declining, outbreaks caused by Clostridium difficile, a known pathobiont, are a growing problem afflicting patients in hospitals and long-term healthcare facilities. C. difficile infection (CDI) is believed to result from gastrointestinal dysbiosis, i.e., the disruption of the resident microbiota. Antibiotics treatment kills most bacteria in the GI tract that usually control C. difficile. In this altered environment, C. difficile replicate and produce toxins that attack the lining of the intestine, causing symptoms ranging from diarrhea to life-threatening inflammation and bleeding of the lining of the colon. According to the Centers for Disease Control and Prevention (CDC), C. difficile alone is linked to the deaths of 14,000 people a year in the United States. In hospitals, C. difficile spores shed in feces are transferred to patients and surfaces mainly via the hands of healthcare personnel who have touched a contaminated surface or item. An effective treatment against recurrent C. difficile infection is not widely available. Paradoxically, the primary treatment for C. difficile infection is the administration of more antibiotics, with about 20% of patients having recurrences within a month, and many of those have repeated attacks.
An unorthodox, alternate procedure, fecal microbiota transplantation (FMT), in which feces from one “donor” is infused into a patient's intestines, is proving to be far more effective than antibiotics at treating recurrent GI infections. By restoring disturbances to the microbial equilibrium, an infusion of feces from healthy donors appears to keep harmful bacteria, such as C. difficile, at bay, eradicating illness even in patients who have suffered repeated, debilitating bouts. In a small Dutch study at the University of Amsterdam, 15 of 16 patients with recurrent C. difficile infection were cured with duodenal infusion of donor feces, compared to only 27% of patients given a 2-week regimen of the antibiotic vancomycin (van Nood, Els et al. (2013)). It was shown that infusion of donor feces resulted in improvement in the microbial diversity in the patient's GI tract and this diversity persisted over time. Recently, Song et al. (2013) confirmed previous reports that a reduction in microbiota diversity and richness in fecal samples from recurrent C. difficile infection (RCDI) patients was restored after FMT to become similar to that of a healthy donor. In this longitudinal study, FMT predominantly affected Firmicutes and Proteobacteria, and the fecal microbiota continued to change in post-FMT patients for at least 16 weeks.
Importantly, the exact mechanism of action responsible for the success of FMT to treat RCDI remains unknown and there is no clinically validated set of parameters to define a suitable donor or ideal donor microbiota. An easy and effective means to collect feces samples in the field and snap-shot the sampled microbiome in a composition at ambient temperature from large numbers of individuals, both healthy donors and RCDI patients, at multiple time points is needed to map the ‘core’ microbiome found in the GI tract of healthy individuals in a population, upon which can be overlaid the changing microbiome of RCDI patients. Ultimately, RCDI patients in the future will be treated, not with antibiotics, but with customized probiotics (a preparation/supplement containing live bacteria that is taken orally to restore beneficial bacteria to the body) and prebiotics (non-digestible food components, such as oligosaccharides, that promote the activity of target selected groups of the GI microflora) or synbiotics (synergistic combinations of probiotics and prebiotics) to return their microbiome to a healthy state.
To avoid the risk of introducing unidentified, potentially harmful microbes, some hospitals are starting to build self-banking systems. A patient's feces can be banked to use in the future as an antidote against possible infection with hospital-acquired “super bugs.” Using the patient's own feces for transplantation greatly reduces the risk of introducing harmful microbes and avoids time-consuming and costly screening of feces from unrelated donors for transmissible diseases. Unfortunately, it appears the “ecosystem” of certain people, however, makes them more susceptible to illness than others. Hence, a possible drawback associated with reintroducing a patient's own feces is that it may only provide short-term benefits and not cure them of detrimental microbes, such as C. difficile. In time, microbiome research may lead to the identification of ‘core’ or ‘keystone’ bacterial species that help to define human health and then develop personalized “bacteriotherapy,” consisting of fully characterized, beneficial bacterial “cocktails,” to supplant this crude method of transplanting “raw” feces. In fact, probiotics therapies have now been proposed for a large variety of gut-related disorders such as IBD and inflammatory bowel syndrome. Fundamentally, researchers and clinicians attempting to characterize all species of a donor's microbiota, identify diagnostic markers to predict susceptibility to disease, and ultimately provide ‘personalized’ health care, need to be confident that the fecal samples being tested provide a true representation or “snap-shot” of the donor's microbiome in vivo, not a ‘degraded’ or artificial representation of the microbial community. Hence, an effective means to immediately capture and stabilize or snap-shot the microbiome of feces at the point of collection is critical.
Colorectal cancer (CRC) has the highest cancer mortality rates in Europe and the United States. It is known that CRC is highly curable (>90%) if detected in its early stages, making early cancer screening a valuable asset. A number of sensitive examination methods have been devised over the years to detect cancer, such as double-contrast barium enema, colonoscopy, and flexible sigmoidoscopy. However, the financial costs, infrastructure, and manpower requirements associated with these procedures present formidable obstacles, not to mention being uncomfortable and invasive for the patient. In addition to costs, the low-throughput nature of these examination methods impedes their implementation for nationwide primary screening.
Presently, another method to screen for colorectal cancer is the fecal occult blood test (FOBT). This test detects the presence of haemoglobin in feces samples to determine the presence or absence of bleeding in GI tract, as an indirect predictor of CRC. While this test is not expensive, its sensitivity and positive predictive value is very low and the incidence of false-positives is high. Therefore, a sensitive, reliable, cost-effective, scalable method is in great need for both diagnosis of disease in at-risk and/or symptomatic individuals, as well as for routine diagnostic screening of the asymptomatic population. Ideally, an individual would routinely collect and stabilize a portion of their feces in the privacy of their home and then mail it to a testing facility to be screened for CRC and other diseases.
It is already accepted that direct detection and examination of tumour cells sloughed into the colonic lumen and recovered from feces is a more positive predictor of colorectal cancer than occult blood. However, the “target” or mutant human DNA, indicative of cancer or other diseases, is usually present in the biological sample at low frequency (e.g. 1% of total human DNA for CRC), often against a high background of wild-type DNA (e.g. bacterial DNA and human DNA from normal colon cells), and exposed to endogenous human DNases (e.g. deoxyribonuclease I) and/or bacterial nucleases (e.g. Micrococcal nuclease). In this complex specimen, what little “target” human DNA that exists in a fecal sample may be rapidly degraded by nucleases and environmental conditions before it even reaches the laboratory, negatively impacting clinical sensitivity of diagnostic tests. In addition to the abundance of nucleases, anaerobic bacteria, constituting over 99% of bacteria in the gut, become exposed to air as soon as feces are eliminated from the digestive tract. Air, specifically oxygen, is a toxic environment to anaerobic bacteria killing 50% within 4-5 minutes and 95-97% of anaerobes after only 20 minutes (Brusa et al., 1989). Again, acquiring a representative view or “snap-shot” of the entire microbiome and human DNA in feces is a challenge considering most fecal samples are collected at home, not in a laboratory or healthcare facility.
It is imperative to stabilize total nucleic acid in biological samples such that it does not degrade during sample handling, transport and storage. To minimize degradation of nucleic acid in biological samples, it is standard practice to transport whole samples or portions thereof on dry ice (−78° C.) to centralized testing facilities where it is either thawed and processed immediately or kept frozen in storage (−80° C. to −20° C.). The costs, logistics and infrastructure needed to ensure collected samples are frozen immediately, kept frozen during transport to testing facilities, and stored under optimal conditions prior to analysis, poses significant challenges and risks, especially in large-scale and population-based screening applications. It can be even more challenging to provide ‘representative’ samples for decentralized sample analysis and still retain maximum sample integrity. It is highly desirable to develop a more robust and standardized sample-handling method and composition that captures and maintains a true representation of each sample's nucleic acid profile.
The study of the relationship between the microbiome and its human host in health and disease relies on the identification and monitoring the microbial communities over a period of time. Recent discoveries demonstrate the utility of these microbial profiles as biomarkers with prognostic and diagnostic value. It is becoming evident in the literature that due to the dynamic nature of the gut microbiome, repeated sampling of large populations over time is essential to the development of such biomarkers. These studies, known as Microbiome-Wide Association Studies (MWAS) are challenged by low donor compliance, unreliable self-collection of biological samples, high cost and cumbersome shipping and handling procedures.
Current methods for feces sampling and microbiota analysis involve the transport of specimens under conditions that have the potential to expose samples to temperatures incompatible with microbiome stabilization. Failure to properly stabilize the microbiome during sample collection, transport, processing and analysis risks obscuring the biological and clinical meaning of the microbiome profile. Consequently, proper pre-analytical procedures are necessary to ensure the best possible representation of the in vivo microbiome profile.
There is a need for compositions and methods for stabilizing nucleic acids, in particular both human and microbial DNA, in complex biological samples such as feces, during transport and storage at ambient temperatures.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.